Bovine Paratuberculosis, commonly referred to as Johne's disease, is a contagious bacterial disease estimated to be present in 68.1% of U.S. dairy herds (APHIS 2008) and results in annual losses exceeding U.S. $200 million (Ott et al. 1999). The bacterium Mycobacterium avium subspecies paratuberculosis (Map) is responsible for Johne's disease and causes reduced milk production, reproductive failure, weight loss, and eventual death. Johne's disease is not treatable, and vaccination against Map has been largely unsuccessful.
Because effective diagnosis, treatment and management strategies for Johne's disease are difficult and expensive to implement, a complementary strategy to reduce the susceptibility of cattle to Johne's disease is through improving host genetics through selective breeding. Susceptibility to Map infection in cattle is heritable, with heritability estimated to range from 0.06 to 0.183 (Koets et al. 2000; Mortensen et al. 2004; Gonda et al. 2006). Breed-specific differences in susceptibility have also been reported, with Jersey and Shorthorn breeds being more susceptible than Holstein (Cetinkaya et al. 1997; Jakobsen et al. 2000). These results indicate that susceptibility to Map infection is at least partially determined by inherent genetic factors, and that breeding for increased resistance to Johne's disease may be possible.
The identification of animals with genetic susceptibility, resistance, or tolerance to infection by Map would provide mechanisms to reduce the incidence of Johne's disease. Further, a reduction of Map in the environment may also be beneficial to humans, as the presence of Map has also been implicated in the etiology of Crohn's disease (Bentley et al. 2008). It has been demonstrated that the genetic background of an animal plays a role in its resistance to Johne's disease (Settles et al., 2009). Cattle that are exposed to Mycobacterium avium subspecies paratuberulosis (Map) respond by either resisting or clearing the infection or becoming chronically infected with varying levels of disease severity.
Resistance or susceptibility to Map infection has been shown to have a hereditary component in cattle and mice (Koets et al. 2000; Mortensen et al. 2004; Gonda et al. 2006; Hinger et al. 2008), with estimates ranging from 0.06 to 0.102. However, attempts to locate genetic loci associated with resistance to paratuberculosis have had limited success. Gonda et al. (2007) found evidence for a quantitative trait locus (QTL) on Bos taurus chromosome 20 (BTA20) associated with paratuberculosis susceptibility. Hinger et al. (2007) investigated the association with paratuberculosis of 8 microsatellites located in or near Map susceptibility candidate genes in 1,179 (594 positive) German Holstein cows. However, none showed any significant associations. While Map susceptibility genes have yet to be identified in the bovine, Reddacliff et al. (2005) found an association of one microsatellite allele in SLC11A1 (formerly NRAMP1) with Map resistance in sheep.
Resistance to Map has been shown in mice to be associated with the Bcg gene or nramp1 which encodes the natural resistance-associated macrophage protein (Frelier et al. 1990; Skamene, 1989; Skamene et al. 1982). C57/B6 and BALB/c mice have the susceptible allele of Bcg and are susceptible to Map infections, while the C3H/HeJ strain is resistant to Map (Veazey et al. 1995a; Veazey et al. 1995b; Tanaka et al. 1994; Chiodini et al. 1993; Chandler 1962; Tanaka et al. 1994). In cattle, Map-susceptible Holstein sire lines have been found to be infected twice as often as resistant lines (Gonda et al. 2006). Heritability studies have been conducted on the presence or absence of disease based on postmortem tissue, ELISA and combined ELISA-fecal culture tests. In a Dutch study, the heritability of paratuberculosis infection was evaluated among vaccinated and unvaccinated animals based on findings from postmortem examinations (Koets et al. 2000). A heritability of 0.09, 0.01 and 0.06 was found for vaccinated, unvaccinated and all cows, respectively. A second study estimated the heritability of antibody response using a bivariate model with daily milk yield and optical density values from milk ELISAs (Mortensen et al. 2004). Mortensen and coworkers (2004) estimated the heritability to be 0.102 with the bivariate model and 0.091 when a sire model was used. Gonda and colleagues (2006) estimated the heritability of Johne's disease to be 0.153 based on fecal culture diagnostic testing, 0.159 based on ELISA and 0.102 from the combined antibody and fecal culture tests. We (Zanella et al. 2008) estimated the heritability of tolerance to Johne's disease to be 0.09.
Limited investigations have been conducted to identify loci associated with Johne's disease. Using a candidate gene approach, Taylor and colleagues (2006) evaluated the allele frequencies of a functional candidate gene, CARD15, in 30 unrelated unaffected animals and 11 affected animals without finding evidence for an association. Hinger et al. (2007) also investigated the association with Johne's disease utilizing 8 microsatellite genetic markers located in or near Map susceptibility candidate genes in 1,179 (594 positive) German Holstein cows, but none of the microsatellites revealed any associations. Reddacliff et al. (2005) found an association of one microsatellite allele in SLC11A1 (formerly NRAMP1) with Map resistance in sheep.
Gonda and coworkers (2006) undertook a genome-wide linkage study using ELISA, fecal culture or both to diagnose infected animals. In this study, microsatellites were used to genotype three half-sib families. The number of informative (useful) markers ranged from 151-176 within the three families. Genotypes of “positive” and “negative” animals were pooled and allele frequencies were estimated. Eight chromosomal regions were associated with the pooled samples (bovine chromosomes 7, 10, 12, 14, 15, 18, 20 and 25). The eight chromosomal regions associated with Map infection in pooled genotypes were further tested. Individual genotypes of the daughters were determined for 3-5 microsatellites within 15 cM (an estimated 15 million base pairs) of the markers identified in the pooled samples. Subsequently, only chromosome 20 was found to be linked (P=0.0319) in a chromosome-wide analysis in one of the sire families.
Several studies have addressed the identification of genetic loci associated with Map susceptibility by testing candidate genes, by genome-wide linkage or association studies. Polymorphisms in functional candidate genes, SLC11A1 (Pinedo et al. 2009a), TLR1, 2 and 4, (Mucha et al. 2009), CARD15 (Pinedo et al. 2009b), PGLYRP1 (Pant et al. 2010), IL12R (Pant et al. 2011), and IL10Ra (Verchoor et al. 2010) have been reported to be associated with susceptibility to Map infection in cattle. Genome wide linkage analysis provided evidence for a locus for Map susceptibility on BTA20 (Gonda et al. 2007). Recently, several genome wide association studies using a single nucleotide polymorphism (SNP) panel (the Illumina BovineSNP50 BeadChip) identified regions on different chromosomes that are significantly associated with Map infection (Settles et al. 2009; Minozzi et al. 2010; Kirkpatrick et al. 2010; van Hulzen et al. 2011; Minozzi et al. 2012). However, none of these publications present evidence for strong functional candidate genes associated with Johne's disease in these chromosomal regions.
Previous studies have identified several loci associated with Map tissue infection on BTA3 (Settles et al. 2009, Neibergs et al. 2010). The region associated with tissue infection was further refined to a 10.6 kb region (Zanella et al. 2011).
Current management practices are to cull cows after either testing positive for Map or exhibiting clinical signs of the disease. However, clinical signs of Map infection may be delayed as long as four to five years after the initial exposure. Current diagnostic testing has a limited sensitivity for detecting the presence of Map in pre-clinical animals, at which time they are likely to be spreading Map to other animals in the herd through fecal contamination of the environment, food, and water. The low overall sensitivity of ELISA (7-35%) and fecal (38-65%) diagnostic tests and the long incubation period of the disease present major roadblocks to the control of Johne's disease (Whitlock et al. 2000; McKenna et al. 2005; Collins et al. 2006).
Identifying the mutations associated with Map tissue infection will not only provide the opportunity to better understand the genes and gene regulatory elements that are associated with the first event in the pathogenic process that culminates in Johne's disease, but will provide compositions and methods for diagnosis of susceptibility, resistance and/or tolerance to infection by Mycobacterium avium subspecies paratuberculosis (Map).